luteolin improves cardiac dysfunction in heart failure rats by regulating sarcoplasmic reticulum ca2 atpase 2a

www.nature.com/scientificreports OPEN received: 10 December 2015 accepted: 15 December 2016 Published: 23 January 2017 Luteolin improves cardiac dysfunction in heart failure rats by regulating sarcoplasmic reticulum Ca2+-ATPase 2a Wenjing Hu1,*, Tongda Xu2,*, Pei Wu1,*, Defeng Pan1, Junhong Chen2, Jing Chen2, Buchun Zhang1, Hong Zhu2 & Dongye Li1 We previously found that luteolin (Lut) appeared to improve the contractility of cardiomyocytes during ischemia/reperfusion in rats The enhancement was associated with the alteration in sarcoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) This finding prompted us to consider if the mechanism worked in heart failure (HF) We studied the regulation of SERCA2a by Lut in failing cardiomyocytes and intact heart of rats Improvement of contractility and the mechanisms centered on SERCA2a were studied in isolated cardiomyocytes and intact heart We found that Lut significantly improved contractility and Ca2+ transients, ameliorated expression, activity and stability of SERCA2a and upregulated expression of small ubiquitin-related modifier (SUMO) 1, which is a newfound SERCA2a regulator Lut also increased phosphorylation of protein kinase B (Akt), phospholaban (PLB) and sumoylation of SERCA2a, specificity protein (Sp1) Transcriptions of SUMO1 and SERCA2a were concurrently increased Inhibition of posphatidylinositol kinase/Akt (PI3K/Akt) pathway and SERCA2a activity both markedly abolished Lut-induced benefits in vitro and in vivo Lut upregulated the expression ratio of Bcl-2/Bax, caspase-3/cleaved-Caspase3 Meanwhile, Lut ameliorated the myocardium fibrosis of HF These discoveries provide an important potential therapeutic strategy that Lut targeted SERCA2a SUMOylation related to PI3K/Akt-mediated regulations on rescuing the dysfunction of HF Heart failure (HF) is a complex syndrome that results from the deterioration of the cardiac structure and function, characterized by the impaired ability of the ventricle to fill with or eject blood1 It is an ultimate common pathway that begins with diverse etiologies, such as hypertension, ischemia, tachycardia, infection, metabolic disorder, and cardiomyopathy, and develops with continual activation of the renin-angiotensin and sympathetic nervous systems The incidence, prevalence and economic burden of HF are now steadily increasing due to the aging of the population and transition of acute cardiac problems into chronic disorders Abnormality Ca2+ homeostasis is a universal characteristic of human and experimental HF2 Ca2+ homeostasis is directly modulated by four proteins: L-type Ca2+ channel and Na+/Ca2+ exchanger (NCX) in cell membrane, Ca2+-ATPase and ryanodine receptor type in sarcoplasmic reticulum (SR)3 Any abnormality of the expression or activity of the Ca2+ handling proteins mentioned above leads to alterations in cardiac contractility Sarcoplasmic reticulum Ca2+-ATPase 2a (SERCA2a), a principal cardiac form of SERCA, is important in controlling excitation/contraction coupling SERCA2a’s role in HF has been extensively studied in animal models and human, which have shown that SERCA expression and activity are reduced in failing myocardium4 Genetic treatments show that reduction in SERCA2a level results in impaired intracellular Ca2+ homeostasis and reduces both systolic and diastolic function5,6 These results indicate that modulation of SERCA2a is a possible means of regulating cardiomyocytes contractility in HF Multiple mechanisms participate in the regulation of SERCA2a function, both its activity and expression Phospholaban (PLB) is a well-known major regulator of SERCA2a activity Binding of PLB to SERCA2a inhibits the pump’s affinity for Ca2+, whereas phosphorylation of PLB suppresses this inhibition4,7 SERCA2a activity Institute of Cardiovascular Disease Research, Xuzhou Medical University, Xuzhou, Jiangsu, 221002, China Department of Cardiology, Affiliated Hospital of Xuzhou Medical University, Xuzhou, Jiangsu, 221006, China *These authors contributed equally to this work Correspondence and requests for materials should be addressed to D.L (email: dongyeli@medmail.com.cn) or H.Z (email: hongzhumao@sohu.com) Scientific Reports | 7:41017 | DOI: 10.1038/srep41017 www.nature.com/scientificreports/ could also be modulated by post-translational modifications, such as glutathiolation8 and nitration9 Kho et al showed that sumoylation was a critical post-translational modification in regulating SERCA2a function10 Sumoylation is a post-translational modification that can be accomplished by reversibly binding the small ubiquitin-related modifier (SUMO) Sumoylation alters the functional activity of targets by regulating protein stability, protein-DNA binding activity, protein–protein interaction and nucleo-cytoplasmic translocation11 Kho et al found that sumoylation of SERCA2a was significantly reduced in cardiac tissue of HF patients By means of gene therapy, they proved that sumoylation increased the intrinsic activity of SERCA2a ATPase in failing cardiomyocytes, as well as prolonging the life time of SERCA2a Sumoylation of myocardial targets other than SERCA2a might also contribute to the reversal of cardiac dysfunction12 A study by Tilemann et al suggested that sumoylation of Sp1, a recognized transcription factor of SERCA2a in myocardium, resulted in an increase of SERCA2a transcription in the failing heart Therefore, sumoylation may be an effective target for therapy in HF Luteolin (Lut), a widely existing flavonoid in Chinese herbal medicine, enhances the contractility of cardiomyocytes during ischemia/reperfusion (I/R)13,14, which is mostly related to alteration of SERCA2a function Our previous study also confirmed that Lut could decrease serum BNP level and partially reverse ventricular remodeling, finally improve cardiac function of HF rats15 In 2007, the clinical trial of SERCA2a gene therapy has been initiated, which was called Calcium Up-regulation by Percutaneous Administration of Gene Therapy in Cardiac Disease (CUPID)16 As a first phase 1/2 clinical trial, CUPID got a positive result, CUPID2 were followed conducted in 201217 However, results of CUPID2 trial turned out to be negative; revealing no improvement in recurrent and terminal events in patients received AAV1/ SERCA2a, compared to patients with placebo18 Reasons for the negatives results of CUPID2 were still not clear We would discuss the reasons in subsequent section Furthermore, most findings of the improvement in cardiac contractile function are attributed to increase SERCA2a protein or augmented PLB phosphorylation, further mechanisms of the regulation have not been fully elucidated Our another study showed that Lut had cardioprotection against I/R injury by improving the I/R-induced decrease in SERCA2a activity partially via the PI3K/Akt signaling pathway in vivo19 Therefore in the present study, we expected to explore whether, and if so, how Lut enhances the contractility of failing cardiomyocytes and intact heart in rats Results Rats’ survival status and general condition.  The 12 weeks later, all rats in Sham groups were alive 72 rats in AAC group were alive and the rest were died Among the 17 died AAC rats, died in the first four weeks, none died in the second four weeks, and died in the last four weeks It was discovered by anatomy of the dead rats that the main cause of the death in the first four weeks was acute HF The causes of the death in the last four weeks were dyspnea and hydrothorax The 12 weeks later, when compared with their counterparts in sham group, the survival rats in the AAC group were less active and ate less The respiratory rate of these rats increased Echocardiography demonstrated the definitely expanded left ventricle and decreased cardiac function of AAC rats induced by pressure overload.  Echocardiography on rats subjected to 12 weeks of pressure overload detected the occurrence of HF In the AAC group, compared with themselves at 0 w, their left ventricles apparently expanded 12 weeks after surgery More specifically, left ventricular internal diameters of end-diastole (LVIDd) as well as left ventricular internal diameters of end-systole (LVIDs) of 12 w AAC rats were significantly increased (LVIDd: 0.455 ± 0.032 vs 0.783 ± 0.036, p